Method for evaluating production regulator of antigenic antibody targeting type 2 ryanodine receptor

Abstract

To provide: a method for evaluating a production regulator of antigenic antibodies, using a model mouse in which the production of the antigenic antibodies is inhibited; and a method for evaluating adjuvant using the model mouse.SOLUTION: A method for evaluating a production regulator of antigenic antibodies comprises a step of preparing a group of type 2 ryanodine receptor stabilizing mutant mice in which valine at position 3599 in an amino acid sequence described in SEQ ID NO: 1 is substituted with lysine, a group of drug-administered mice which has been administered with an antigenic antibody production regulator targeting the type 2 ryanodine receptor to be evaluated, and which does not have the type 2 ryanodine-receptor stabilizing mutation, and a group of control mice which has not been administered with the antigenic antibody production regulator, and which does not have the type 2 ryanodine receptor-stabilizing mutation, and measuring a production amount of antigenic antibodies in the above three groups of mice.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for evaluating a production regulator of an antigenic antibody targeting a type 2 ryanodine receptor to be evaluated, and a method for evaluating the action of an adjuvant to be evaluated.

Background Art

[0002] Arthritis is one of the physical symptoms with a high incidence among the symptoms of autoimmune diseases. Rheumatoid arthritis is a typical autoimmune disease that causes arthritis, with a prevalence of about 0.5% and the number of patients in Japan reaching about 700,000. Although the functions of the immune system due to lifestyle habits, genetic factors, infections, etc. are involved, the detailed pathogenesis is unknown. As a treatment method for autoimmune diseases, drugs having an immunosuppressive effect such as methotrexate (MTX) and biological agents are widely used. However, these treatment methods have problems such as side effects including the onset of infectious diseases due to increased susceptibility to infection associated with immunosuppression, for example, pneumonia and urinary tract infections.

[0003] In recent years, it has been reported that endoplasmic reticulum (ER) stress is involved in one of the causes of the onset of rheumatoid arthritis. Many molecular chaperones in the endoplasmic reticulum are Ca 2+ -dependent, and when the Ca 2+ concentration in the endoplasmic reticulum is not maintained, the function of the endoplasmic reticulum is significantly reduced, and it is known that endoplasmic reticulum stress occurs. However, the mechanism of the onset of rheumatoid arthritis due to endoplasmic reticulum stress has not been fully elucidated.

[0004] So far, the present inventors have focused on the type 2 ryanodine receptor (RyR2) present in the membrane form of the endoplasmic reticulum in the course of studying endoplasmic reticulum stress. Then, by mutating the calmodulin (CaM) binding domain of the type 2 ryanodine receptor in the myocardium, a RyR2 V3599K KI mouse with enhanced affinity of CaM for RyR2 was produced (see Non-Patent Document 1). It has been reported that this RyR2 V3599K KI mouse does not cause cardiac hypertrophy even in the transverse aortic constriction (TAC) model. In this mouse, Ca 2+It has also been reported that suppression of endoplasmic reticulum stress by leakage suppression can suppress the onset of Alzheimer's disease (see Non-Patent Document 2). Furthermore, dantrolene, which is known to act on type 1, type 2, and type 3 ryanodine receptors to suppress the release of Ca 2+ from the endoplasmic reticulum, has also been reported to suppress the production of autoantibodies and prevent arthritis (see Non-Patent Document 3).

[0005] In addition, a method for treating or suppressing the progression of T cell-mediated autoimmune diseases such as multiple sclerosis by a ryanodine receptor inhibitor has been disclosed (see Patent Document 1). In this document, attention is paid to the intracellular calcium signal transduction and calcium-dependent functions of activated T cells by inhibiting type 1 and type 3 ryanodine receptors, and the involvement of type 2 ryanodine receptor has been considered to be scarce.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] In conventional arthritis drugs, side effects due to excessive immunosuppressive effects are a problem. Therefore, there has been a demand for a drug that has less excessive immunosuppressive effects and can suppress autoimmune diseases in which autoantibodies are produced. However, there was no animal model for suppressing the production of antigenic antibodies, and even if a candidate molecular target drug was found, it was difficult to evaluate its effect. On the other hand, regarding the production of antibodies used as pharmaceuticals and experimental reagents such as vaccines, adjuvants are widely used to increase the production amount, but a method for developing a more potent adjuvant has been demanded. Therefore, an object of the present invention is to provide a method for evaluating a drug for regulating the production of antigenic antibodies using a model mouse in which the production of antigenic antibodies is suppressed, and a method for evaluating the adjuvant effect using the above model mouse.

Means for Solving the Problems

[0009] As a result of intensive studies to solve the above problems, the present inventors prepared mutant mice in which only the type 2 ryanodine receptor, which exists in three types, types 1 to 3, was stabilized, and found that the mutant mice had an effect of suppressing the production of antigenic antibodies. Furthermore, it was confirmed that the mutant mice have an immunomodulatory effect that mainly acts on abnormal immune cells, rather than an immunosuppressive effect that acts on the entire immune cells. Therefore, by using the mutant mice, it was found to be useful for evaluating a production regulator of antigenic antibodies and evaluating an adjuvant effect, and the present invention was completed.

[0010] That is, the present invention is as follows. 〔1〕A group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, A group of drug-administered mice without the above-described type 2 ryanodine receptor stabilization mutation, to which a production regulator of an antigenic antibody targeting the type 2 ryanodine receptor to be evaluated is administered, and A control mouse group without the above-described type 2 ryanodine receptor stabilization mutation, to which the above-described production regulator of an antigenic antibody is not administered, is prepared, A method for evaluating the production regulator of the antigenic antibody, comprising a step of measuring the production amount of the antigenic antibody in the above three mouse groups. 〔2〕The evaluation method according to the above 〔1〕, wherein the production regulator of the antigenic antibody is a molecular target drug for allergic diseases or a molecular target drug for autoimmune diseases targeting the type 2 ryanodine receptor. 〔3〕A group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, and a control mouse group without the above-described type 2 ryanodine receptor stabilization mutation are prepared, and allergic disease or autoimmune disease model mice derived from each mouse group are produced, In an allergic disease or autoimmune disease model mouse derived from a group of type 2 ryanodine receptor-stabilized mutant mice, when the onset of the disease is suppressed and the production of antigenic antibodies is decreased as compared with an allergic disease or autoimmune disease model mouse derived from a control mouse group without the type 2 ryanodine receptor-stabilized mutation, the allergic disease or autoimmune disease comprises a step of evaluating that the efficacy of an antigen-antibody production regulator targeting the type 2 ryanodine receptor can be expected. A group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine A drug-administered mouse group without the type 2 ryanodine receptor-stabilized mutation, to which an antigen-antibody production regulator targeting the type 2 ryanodine receptor to be evaluated is administered, and A control mouse group without the type 2 ryanodine receptor-stabilized mutation, to which the antigen-antibody production regulator is not administered. After sensitizing each of the three mouse groups with an antigen that induces the allergic disease or autoimmune disease, for which the efficacy of the antigen-antibody production regulator targeting the type 2 ryanodine receptor has been evaluated, to produce the allergic disease or autoimmune disease model mouse, the production amount of antigenic antibodies in each model mouse group is measured. The method according to [1] or [2] above, characterized by the above. 〔4〕The method according to [1] or [2] above, characterized in that the production amount of antigenic antibodies in the drug-administered mouse group without the type 2 ryanodine receptor-stabilized mutation is measured more than 2 months after administration of the antigen-antibody production regulator targeting the type 2 ryanodine receptor to be evaluated. 〔5〕A method for evaluating the adjuvant effect, comprising a step of administering an antigen and an adjuvant to be evaluated to a group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine, and measuring the production amount of antigenic antibodies in the mouse group. 〔6〕Furthermore, measure the production amount of antigenic antibodies in the adjuvant-administered mouse group without the type 2 ryanodine receptor stabilization mutation to which the above antigen and the adjuvant to be evaluated are administered and / or the control mouse group without the type 2 ryanodine receptor stabilization mutation that is not administered the above antigen and the adjuvant to be evaluated, and evaluate the adjuvant effect by the method described in the above 〔5〕.

[0011] Furthermore, another aspect 1 of the present invention is as follows. (i) A group of type 2 ryanodine receptor stabilization mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine, Prepare a control mouse group without the type 2 ryanodine receptor stabilization mutation, Sensitize the above two mouse groups with an antigen that induces a predetermined disease to prepare a predetermined disease model mouse, Compare each disease model mouse, and when the onset of a predetermined disease is suppressed and the antibody production against the sensitized antigen is reduced in the disease model mouse derived from the type 2 ryanodine receptor stabilization mutant mouse group compared to the disease model mouse derived from the control mouse group, the disease model mouse derived from the type 2 ryanodine receptor stabilization mutant mouse group is evaluated as a disease model mouse in which an antigen-antibody production regulating agent targeting the type 2 ryanodine receptor is effective.

[0012] Furthermore, another aspect 2 of the present invention is as follows. (ii) A method for evaluating the drug efficacy of a drug to be evaluated or the side effects caused by the drug, which comprises administering to a type 2 ryanodine receptor stabilization mutant mouse in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine a drug to be evaluated that is likely to cause at least one side effect selected from anaphylaxis, rejection reaction, and respiratory failure due to antigenic antibodies, and evaluating the drug efficacy or the side effects caused by the drug after at least 2 months have elapsed since the administration.

Effects of the Invention

[0013] By the method for evaluating an agent for regulating the production of an antigenic antibody of the present invention, it becomes possible to evaluate the regulation of the production of an antigenic antibody in an agent for regulating the production of an antigenic antibody to be evaluated. Furthermore, by the method for evaluating an adjuvant action of the present invention, it becomes possible to evaluate the adjuvant action.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0015] The contents described in all patent documents and non-patent documents cited in this specification are hereby incorporated by reference in their entirety into this specification.

[0016] The method for evaluating a production regulator of an antigenic antibody of the present invention comprises a step of preparing a group of type 2 ryanodine receptor-stabilized mutant mice (hereinafter also referred to as "mutant mouse group") in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine, a group of drug-administered mice without the above-described type 2 ryanodine receptor stabilization mutation (hereinafter also referred to as "drug-administered non-mutant mouse group") to which a production regulator of an antigenic antibody targeting the type 2 ryanodine receptor to be evaluated is administered, and a group of mice without the above-described type 2 ryanodine receptor stabilization mutation without administration of the above-described production regulator of an antigenic antibody (hereinafter also referred to as "control mouse group"), and measuring the production amount of the antigenic antibody in the above three mouse groups. The method is not particularly limited as long as it is a method for evaluating the production regulator of the antigenic antibody, and is hereinafter also referred to as "the method for evaluating the production regulator of the antigenic antibody in the present case". Further, the method for evaluating the adjuvant action of the present invention is not particularly limited as long as it comprises a step of administering an antigen and an adjuvant to be evaluated to a group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine, and measuring the production amount of the antigenic antibody in the above mouse group, and is hereinafter also referred to as "the method for evaluating the adjuvant action in the present case".

[0017] (Type 2 ryanodine receptor) Intracellularly, the type 2 ryanodine receptor (RyR2), which is a tetrameric protein, exists on the endoplasmic reticulum membrane. When oxidative stress or gene mutation is applied to the type 2 ryanodine receptor, calmodulin (CaM) is dissociated from the type 2 ryanodine receptor. As a result, the tetrameric structure of the type 2 ryanodine receptor is destabilized, and calcium ions (Ca 2+ ) leak out of the endoplasmic reticulum, and the intracellular Ca 2+ concentration increases. The increase in the intracellular Ca 2+ concentration is considered to be the cause of malignant hyperthermia, fatal arrhythmia, etc.

[0018] The calmodulin-binding domain of the type 2 ryanodine receptor is residues 3584 to 3603 in the amino acid sequence set forth in SEQ ID NO: 1. Among these, the type 2 ryanodine receptor in which valine (V) at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine (K) has an improved binding affinity of calmodulin for the calmodulin-binding domain. As a result, leakage of calcium ions (Ca 2+ 2+) outside the endoplasmic reticulum is suppressed (see Non-Patent Document 1 above). Therefore, a mutant mouse in which valine (V) at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine (K) to stabilize the type 2 ryanodine receptor serves as a model in which leakage of calcium ions from the endoplasmic reticulum is suppressed by a molecular target drug targeting the type 2 ryanodine receptor.

[0019] (Production of type 2 ryanodine receptor-stabilized mutant mouse) In the present specification, a mouse in which valine (V) at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine (K) to stabilize the type 2 ryanodine receptor (hereinafter, also referred to as "type 2 ryanodine receptor-stabilized mutant mouse" or "V3599K KI mouse") can be produced by using a genome editing technique such as the CRISPER (Clustered Regularly Interspaced Short Palindromic Repeat)-CAS9 system, the CRISPR-CAS3 system, or TALEN to substitute the 10795-10797th base (the codon corresponding to valine) in the polynucleotide encoding the type 2 ryanodine receptor on the mouse genome set forth in SEQ ID NO: 2 with the codon corresponding to lysine.

[0020] ■ Method for evaluating antigenic antibody production regulator (Antigenic antibody, antigenic antibody production regulator) As used herein, an antigenic antibody refers to an antibody that appears upon sensitization with an antigen and binds to the sensitized antigen. This antigenic antibody is an antibody produced by antigen presentation from antigen-presenting cells upon sensitization with an antigen such as an external substance or a self-antigen, and as a result, activation of B cells. Examples of the types of antigenic antibodies include IgG or IgE antibodies. The method of sensitizing the antigen is not particularly limited, and examples thereof include administering the antigen subcutaneously or intramuscularly together with a known adjuvant, and inhaling the antigen after intraperitoneal administration together with a known adjuvant.

[0021] Examples of the agent for regulating the production of an antigenic antibody targeting the above-mentioned ryanodine receptor type 2 include a molecular target drug for treating allergic diseases targeting the ryanodine receptor type 2 and a molecular target drug for autoimmune diseases targeting the ryanodine receptor type 2.

[0022] The above-mentioned allergic diseases are preferably type I allergies involving IgE, type II or type III allergies involving IgG. Examples of type I allergies include food allergies, allergic rhinitis, bronchial asthma, atopic dermatitis, drug allergies, hay fever, allergic conjunctivitis, urticaria, anaphylactic shock, etc. Examples of food allergies include allergies to eggs, milk, soybeans, wheat, peanuts, nuts, fish, shellfish, etc. An example of type II allergy is drug-induced hemolytic anemia. An example of type III allergy is hypersensitivity pneumonitis.

[0023] Examples of the autoimmune diseases include diseases that cause abnormal autoantibodies due to autoimmune abnormalities. Specifically, they include rheumatoid arthritis; Sjögren's syndrome; systemic lupus erythematosus; mixed connective tissue disease; dermatomyositis; polymyositis; systemic sclerosis; ANCA-associated vasculitis; Goodpasture syndrome; IgG4-related disease; autoimmune hemolytic anemia; idiopathic thrombocytopenic purpura; antiphospholipid antibody syndrome; Graves' disease; Hashimoto's disease; myasthenia gravis; neuromyelitis optica; autoimmune encephalitis; Guillain-Barré syndrome; autoimmune hepatitis; primary biliary cholangitis; pemphigus; pemphigoid; type 1 diabetes, etc.

[0024] Furthermore, the allergic disease or autoimmune disease is preferably an allergic disease or autoimmune disease for which the efficacy of an antigen-antibody production regulator targeting the type 2 ryanodine receptor can be expected. Whether the efficacy of an antigen-antibody production regulator targeting the type 2 ryanodine receptor can be expected for the allergic disease or autoimmune disease can be evaluated by the following method.

[0025] First, prepare a group of type 2 ryanodine receptor-stabilized mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, and a control mouse group without the type 2 ryanodine receptor-stabilized mutation, and produce allergic disease or autoimmune disease model mice derived from each mouse group. Next, in the allergic disease or autoimmune disease model mice derived from the group of type 2 ryanodine receptor-stabilized mutant mice, when the onset of the disease is suppressed and the production of antigenic antibodies is reduced as compared with the allergic disease or autoimmune disease model mice derived from the control mouse group without the type 2 ryanodine receptor-stabilized mutation, it can be evaluated that the allergic disease or autoimmune disease is effective for an antigen-antibody production regulator targeting the type 2 ryanodine receptor.

[0026] (Mouse) In the method for evaluating the production regulator of the antigenic antibody of this case and the method for evaluating the adjuvant action of this case described below, as the strains of mice used in the mutant mouse group, the non-mutant mouse group administered with the drug, and the control mouse group, C57BL / 6 mice, BALB / c mice, DBA1 mice, C3H / He mice, AKR / Ms mice, A / J mice, CT26 mice, 4T1 mice, MAD109 mice, and RENCA mice can be mentioned. In addition, it is preferable to use mice of the same strain for each mouse group.

[0027] The mutant mouse group, the non-mutant mouse group administered with the drug, and the control mouse group may be predetermined disease model mice. Examples of the predetermined disease model mice include autoimmune disease model mice or allergic disease model mice such as arthritis model mice, antigen-antibody induction model mice, autoimmune encephalitis mice, asthma model mice, and anaphylaxis model mice. The above disease models can be prepared by known methods. For example, arthritis model mice can be prepared by mixing type 2 collagen with an adjuvant and subcutaneously injecting it, and allergic disease model mice can be prepared by subcutaneously injecting or intraperitoneally injecting an allergenic substance such as ovalbumin (OVA).

[0028] In addition, as the above disease model mice, allergic disease or autoimmune disease model mice in which the efficacy of the antigen-antibody production regulator targeting the above type 2 ryanodine receptor can be expected may be used. Whether the allergic disease or autoimmune disease model mice can be expected to be effective for the antigen-antibody production regulator targeting the type 2 ryanodine receptor can be evaluated by the following method.

[0029] First, prepare a group of type 2 ryanodine receptor-stabilizing mutant mice in which the 3599th valine in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, and a control mouse group without the type 2 ryanodine receptor-stabilizing mutation, and create allergic disease or autoimmune disease model mice derived from each mouse group. Next, in the allergic disease or autoimmune disease model mice derived from the type 2 ryanodine receptor-stabilizing mutant mouse group, when the onset of the disease is suppressed and the production of antigenic antibodies is reduced as compared with the allergic disease or autoimmune disease model mice derived from the control mouse group without the type 2 ryanodine receptor-stabilizing mutation, the allergic disease or autoimmune disease model mice derived from the control mouse group without the type 2 ryanodine receptor-stabilizing mutation can be evaluated as allergic disease or autoimmune disease model mice in which the efficacy of an antigen-antibody production regulator targeting the type 2 ryanodine receptor can be expected.

[0030] (Method for evaluating antigen-antibody production regulator) In the method for evaluating the antigen-antibody production regulator of the present invention, the production amount of antigenic antibodies in each mouse group can be measured by measuring the amount of antigenic antibodies in the serum or bronchoalveolar lavage fluid of each mouse group. The amount of antigenic antibodies in the serum or bronchoalveolar lavage fluid can be measured by a known antibody amount measurement method such as an enzyme-linked immunosorbent assay (ELISA method), or a commercially available antibody measurement kit may be used. Further, the total IgG antibody amount may be examined together with the antigenic antibody amount. For the drug-administered mouse group without the type 2 ryanodine receptor-stabilizing mutation, the production amount of antigenic antibodies can be measured 3 days, 1 week, 2 weeks, 1 month, 2 months, and 3 months after drug administration.

[0031] Examples of methods for evaluating an antigen-antibody production regulator include the following methods. Prepare the following three mouse groups. 1. A group of V3599K KI mice (mutant mouse group) derived from a predetermined mouse 2. A predetermined group of mice (drug-administered non-mutant mouse group) to which a production regulator of an antigenic antibody to be evaluated is administered 3. A predetermined group of mice that is a non-drug-administered group of a production regulator of an antigenic antibody (control mouse group) A substance that induces a predetermined disease is administered as an antigen to each mouse group to prepare a model mouse of the predetermined disease, and the production amount of IgG or IgE against the substance that induces the predetermined disease administered in the serum is examined. In the mutant mouse group as compared with the control group, the production amount of IgG or IgE against the substance that induces the predetermined disease decreases. When the production amount of the above IgG of the non-administered mutant mouse dissociates from the production amount of the above IgG or IgE of the mutant mouse, it can be determined that the drug to be evaluated does not specifically target the type 2 ryanodine receptor as a molecular target. On the other hand, when the production amount of IgG or IgE of the non-drug-administered mutant mouse is the same as the production amount of IgG or IgE of the mutant mouse, it can be determined that there is a high possibility that the drug to be evaluated specifically targets the type 2 ryanodine receptor as a molecular target.

[0032] Furthermore, the onset of a predetermined disease in a model mouse of the predetermined disease derived from the mutant mouse group and a model mouse of the predetermined disease derived from the control mouse group is compared, and if the onset of the predetermined disease in the model mouse of the predetermined disease derived from the mutant mouse group is suppressed as compared with the model mouse of the predetermined disease derived from the control mouse group, it can be evaluated that the type 2 ryanodine receptor is involved in the predetermined disease.

[0033] ■ Method for evaluating adjuvant action The above mutant mouse group is a model mouse with a low production amount of antigenic antibody. Therefore, an adjuvant that increases the production amount of antigenic antibody using this model mouse can be evaluated as having a high adjuvant action.

[0034] The adjuvant in this specification means a substance that enhances the antibody production ability against an antigen by an immune response, and the adjuvant action means an action that enhances the antibody production ability against an antigen by an immune response.

[0035] The antigen to be administered together with the adjuvant is not particularly limited, and examples thereof include viruses, bacteria, cells, proteins, polysaccharides, etc., and it may be a natural substance or a substance artificially produced by techniques such as genetic recombination.

[0036] As a method (1) for evaluating the adjuvant effect, for example, the following methods can be mentioned. An adjuvant to be evaluated, an existing Freund's adjuvant as a comparison target, and an antigen that induces a predetermined disease are administered to a group of V3599K KI mice (mutant mouse group) derived from a predetermined mouse, and the production amount of IgG against the above antigen is examined. An adjuvant to be evaluated with a higher production amount of IgG against the above antigen than when using the Freund's adjuvant as a comparison target can be evaluated as having a high adjuvant effect.

[0037] Further, as a method (2) for evaluating the adjuvant effect, an adjuvant to be evaluated and an antigen that induces a predetermined disease are administered to a group of V3599K KI mice (mutant mouse group) derived from a predetermined mouse and a group of predetermined mice (control mouse group), and the production amount of IgG against the above antigen in each mouse group is examined. An adjuvant with a production amount of IgG in the mutant mouse group close to the production amount of IgG in the control mouse group can be evaluated as having a high adjuvant effect.

[0038] ■ Evaluation of long-term drug efficacy and side effects The above V3599K KI mice are immunomodulatory mice rather than immunodeficient mice, and also have the characteristic that they are expected to survive longer than immunodeficient mice such as nude mice. Therefore, when a substance that is likely to cause side effects such as anaphylaxis due to an antigenic antibody is administered, the above V3599 mouse K KI (mutant mouse) can evaluate the long-term (for example, 2 months or more) drug efficacy and side effects compared with immunodeficient mice.

[0039] Hereinafter, the present invention will be described more specifically by examples, but the technical scope of the present invention is not limited to these illustrations.

[0040] [Example 1] (Preparation of Ovalbumin (OVA)-IgG Producing Mice) Using ovalbumin as an antigen, OVA-IgG producing mice, which are antigenic antibodies that appear by antigen sensitization, were prepared by the following method.

[0041] First, C57BL / 6-derived V3599K KI mice (produced by commissioning UNITEC) having an internal point mutation in RyR2 (substituting valine (V) at position 3599 of the type 2 ryanodine receptor with lysine (K)) were used using the CRISPR / Cas9 system so as to increase calmodulin (CaM) binding affinity. The above point mutation mutated guanine (G) at position 10795 and thymine (T) at position 10796 in the nucleotide sequence set forth in SEQ ID NO: 2 to adenine (A).

[0042] Next, OVA-IgG producing mice were prepared by the method shown below using C57BL / 6 mice (SLC) and the above V3599K KI mice. First, on Day 0, for each 7- to 10-week-old C57BL / 6 (female) and V3599K KI mouse (female), a reagent (100 μL / mouse) mixed with 50 μL of complete Freund's adjuvant (Cat 231131: BD Biosciences) and 100 μg of OVA (A5503-1G: Sigma-Aldrich) mixed with 50 μL of phosphate-buffered saline was subcutaneously injected into the back of the mouse. Then, on Day 21, for each mouse, a reagent (100 μL / mouse) mixed with 50 μL of incomplete Freund's adjuvant (Cat 263910: BD Biosciences) and 100 μg of OVA (A5503-1G: Sigma-Aldrich) mixed with 50 μL of phosphate-buffered saline was subcutaneously injected into the back of the mouse again. The mice were bred until Day 63 and euthanized on Day 63, and the following analysis (measurement of antibody production amount) was performed. Note that administration of OVA to the mice induces the production of OVA-IgG (IgG antibody against OVA), which is an antigenic antibody against OVA.

[0043] (Evaluation of OVA-IgG level) The levels of mouse OVA-IgG in the sera of OVA-IgG-producing mice derived from C57BL / 6 mice (control mice) and OVA-IgG-producing mice derived from V3599K KI mice, which had been raised until Day 63 by the above method to induce antibody production, were measured by enzyme-linked immunosorbent assay (Mouse Anti-OVA IgG Antibody Assay Kit; catalog Number 3011: Chondrex). The results are shown in Figure 1.

[0044] From the results in Figure 1, the production level of OVA-IgG in OVA-IgG-producing mice derived from V3599K KI mice was significantly suppressed compared with that in OVA-IgG-producing mice derived from control mice. Therefore, it was confirmed that V3599K KI mice have a low ability to produce antigenic antibodies.

[0045] (Evaluation of total IgG level) In addition, the total IgG levels in the sera of OVA-IgG-producing mice derived from control mice and OVA-IgG-producing mice derived from V3599K KI mice, which had been raised until Day 63 by the above method to induce antibody production, were measured by enzyme-linked immunosorbent assay (Mouse Total IgG Antibody Detection Kit; catalog Number 3023: Chondrex). The results are shown in Figure 2.

[0046] From Figure 2, the total IgG level in OVA-IgG-producing mice derived from V3599K KI mice was lower than that in OVA-IgG-producing mice derived from control mice, but the rate of decrease in total IgG level was extremely low compared with the rate of decrease in OVA-IgG level. From this, it is considered that V3599K KI mice have an immunomodulatory effect that mainly acts on abnormal immune cells, rather than an immunosuppressive effect that acts on all immune cells.

[0047] Summarizing the results in Figures 1 and 2 above, it was confirmed that V3599K KI mice are a model specialized in regulating the production of antigenic antibodies among the antibodies in the body.

[0048] [Example 2] (Evaluation method of a production regulator of an antigenic antibody targeting the type 2 ryanodine receptor) Prepare the following three groups of mice. 1. V3599K KI mouse group derived from C57BL / 6 (mutant mouse group) 2. C57BL / 6 mouse group administered with a production regulator of the antigenic antibody to be evaluated (drug-administered non-mutant mouse group) 3. C57BL / 6 mouse group that is a non-drug-administered group for the production regulator of the antigenic antibody (control mouse group) Administer OVA, which is an allergen, as an antigen to each mouse group to prepare allergic disease model mice, and examine the production levels of IgG or IgE against the administered OVA in the serum. In the allergic disease model mice derived from the mutant mouse group compared to those from the control group, the production levels of IgG or IgE against OVA decrease. When the production levels of IgG or IgE against OVA in the allergic disease model mice derived from the drug-administered non-mutant mouse group are dissociated from the production levels of IgG or IgE against OVA in the allergic disease model mice derived from the mutant mouse group, it is determined that the drug to be evaluated does not specifically target the type 2 ryanodine receptor as a molecular target. On the other hand, when the production levels of IgG or IgE against OVA in the allergic disease model mice derived from the drug-administered non-mutant mouse group are similar to the production levels of IgG or IgE against OVA in the allergic disease model mice derived from the mutant mouse group, it is determined that there is a high possibility that the drug to be evaluated specifically targets the type 2 ryanodine receptor as a molecular target.

[0049] Furthermore, compare the onset of allergic diseases in the allergic disease model mice derived from the mutant mouse group and the allergic disease model mice derived from the control mouse group. If the onset of allergic diseases in the allergic disease model mice derived from the mutant mouse group is suppressed compared to that in the allergic disease model mice derived from the control mouse group, it can be evaluated that the type 2 ryanodine receptor is involved in the allergic disease.

[0050] [Example 3] (Method 1 for evaluating the adjuvant effect of the adjuvant to be evaluated) The adjuvant to be evaluated, the existing Freund's adjuvant as a comparison target, and OVA as an antigen are administered to a group of V3599K KI mice (mutant mouse group) derived from C57BL / 6, and the production amount of IgG against OVA is examined. An adjuvant with a higher production amount of IgG than when using the Freund's adjuvant as a comparison target can be evaluated as having a high adjuvant effect.

[0051] [Example 4] (Method 2 for evaluating the adjuvant effect of the adjuvant to be evaluated) The adjuvant to be evaluated and OVA as an antigen are administered to a group of V3599K KI mice (mutant mouse group) derived from C57BL / 6 and a group of C57BL / 6 mice (control mouse group), and the production amount of IgG against OVA is examined. An adjuvant with a production amount of IgG in the mutant mouse group close to the production amount of IgG in the control mouse group can be evaluated as having a high adjuvant effect.

Claims

1. A group of type 2 ryanodine receptor-stabilizing mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, A drug-administered mouse group without the above-described type 2 ryanodine receptor-stabilizing mutation, to which a production regulator of an antigenic antibody targeting the type 2 ryanodine receptor to be evaluated is administered, and A control mouse group without the above-described type 2 ryanodine receptor-stabilizing mutation, to which the above-described production regulator of the antigenic antibody is not administered, is prepared, An evaluation method for the production regulator of the antigenic antibody, comprising a step of measuring the production amount of the antigenic antibody in the above three mouse groups.

2. The evaluation method according to claim 1, wherein the production regulator of the antigenic antibody is a molecular target drug for allergic diseases or a molecular target drug for autoimmune diseases targeting the type 2 ryanodine receptor.

3. A group of type 2 ryanodine receptor-stabilizing mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, and a control mouse group without the above-described type 2 ryanodine receptor-stabilizing mutation are prepared, and allergic disease or autoimmune disease model mice derived from each mouse group are produced. In allergic disease or autoimmune disease model mice derived from the group of type 2 ryanodine receptor-stabilizing mutant mice, when the onset of the disease is suppressed and the production of the antigenic antibody is decreased as compared with allergic disease or autoimmune disease model mice derived from the control mouse group without the above-described type 2 ryanodine receptor-stabilizing mutation, it is evaluated that the above allergic disease or autoimmune disease can be expected to be effective for a production regulator of antigen-antibody targeting the type 2 ryanodine receptor. A group of type 2 ryanodine receptor-stabilizing mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with lysine, A drug-administered mouse group without the above-described type 2 ryanodine receptor-stabilizing mutation, to which a production regulator of an antigenic antibody targeting the type 2 ryanodine receptor to be evaluated is administered, and The antigen that induces the allergic disease or autoimmune disease, which was evaluated as having the potential efficacy of the antigen-antibody production regulator targeting the type 2 ryanodine receptor, was used to sensitize each of the three mouse groups: a control mouse group without administration of the above-mentioned antigen-antibody production regulator and without the type 2 ryanodine receptor stabilizing mutation. After establishing the allergic disease or autoimmune disease model mice, the amount of antigenic antibody produced in each model mouse group was measured. The evaluation method according to claim 1 or 2, characterized by the above.

4. The method according to claim 1 or 2, characterized by measuring the amount of antigenic antibody produced in the drug-administered mouse group without the type 2 ryanodine receptor stabilizing mutation more than two months after administration of the antigen-antibody production regulator targeting the type 2 ryanodine receptor to be evaluated.

5. A method for evaluating the adjuvant effect, comprising administering an antigen and an adjuvant to be evaluated to a group of type 2 ryanodine receptor stabilizing mutant mice in which valine at position 3599 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with lysine, and measuring the amount of antigenic antibody produced in the mouse group.

6. The method according to claim 5, wherein the adjuvant effect is evaluated by measuring the amount of antigenic antibody produced in the adjuvant-administered mouse group without the type 2 ryanodine receptor stabilizing mutation, to which the above antigen and the adjuvant to be evaluated have been further administered, and / or the control mouse group without the type 2 ryanodine receptor stabilizing mutation, to which the above antigen and the adjuvant to be evaluated have not been administered.

Images